Regulation of gingival fibroblast phenotype by periodontal ligament cells in vitro

Abstract Objectives Stem cell transplantation has shown modest effects on periodontal tissue regeneration, and it is still unclear how regenerative effects utilizing this modality are mediated. A greater understanding of the basic interactions between implanted and host cells is needed to improve future strategies. The aims of this study were to investigate the effects of periodontal ligament (PDL) cells on expression of periodontal markers and alkaline phosphatase (ALP) activity of gingival fibroblasts (GF). Materials and Methods Primary human PDL cells were co‐cultured with primary GF cultures either by direct co‐culture with subsequent FACS sorting or indirect co‐culture using transwell cultures and PDL cell conditioned medium. Expression of periodontal markers, asporin, nestin, and periostin, was assessed by qPCR and immunofluorescence staining. Alkaline phosphatase (ALP) expression was assessed by qPCR, histochemical staining, and activity assessed by para‐nitrophenol enzymatic assay. Single cultures of PDL cells and GF were used as controls. The role of Wnt signaling on ALP activity was assessed via Dkk1‐mediated inhibition. Results PDL cells significantly upregulated expression of PDL markers in GF with both direct and indirect co‐culture methods when compared to controls (6.05 vs. 0.73 and 59.48 vs. 17.55 fold change of asporin expression). PDL/GF cell co‐cultures significantly increased ALP activity in GF when compared with single GF cultures. Similar results were obtained when using conditioned medium isolated from PDL cell cultures. Dkk1 caused dose‐dependent reduction in ALP activity of GF cultured in PDL cell conditioned medium. Conclusions PDL cells stimulate expression of periodontal markers and osteogenic capacity of gingival fibroblasts via paracrine signaling which can be partially inhibited with addition of the Wnt antagonist, Dkk1.Further studies are required to identify specific secreted factors responsible for this activity.


| INTRODUC TI ON
There is considerable interest in the possibility of using mesenchymal stem cell (MSC) therapies to promote periodontal regeneration. 1 A number of animal studies have now demonstrated the principle that implantation of either autologous or allogeneic MSC derived from different sources may contribute to the outcome of periodontal regeneration in preclinical models, 2 but this has far only translated modestly in human studies. 3 The presence of high numbers of MSCs within the PDL, as shown by their differentiation potentials, self-renewal, and ability to contribute to periodontal tissue regeneration when implanted into periodontal defects has been formally demonstrated 4 and with greater consistency compared to those derived from other sources. 5 It is also known that in addition to the potential for exogenously applied stem cells to contribute directly to tissue regeneration, they also have properties of immunomodulation, homing, and recruitment of endogenous host cells via paracrine signaling mechanisms. 2 In preclinical models, there is some evidence that periodontal ligamentderived MSC (PDLSC) may contribute directly to new cementum formation, 4 but also that they might stimulate periodontal regeneration indirectly through their interaction with existing endogenous host cells 6,7 Periodontal ligament cells have distinct phenotypic features including the expression of alkaline phosphatase, nestin, asporin, and periostin. [8][9][10][11] Asporin (periodontal ligament associated protein 1, PLAP-1) and periostin are matrix proteins that are particularly highly expressed within the periodontal ligament. Nestin is a neural crestderived cell marker, and its expression in PDL cells reflects their embryological origin. Alkaline phosphatase is also constitutively expressed in the PDL, and its expression is upregulated during osteoblastic differentiation.
A number of studies have demonstrated the presence of MSCs in both healthy and inflamed gingivae, 12,13 which raise the issue of whether these cells might also contribute to periodontal tissue regeneration and might be recruited during regenerative wound healing to contribute to the healing tissues. [14][15][16] Therefore, the aim of study described here was to investigate the hypothesis that PDLSC may induce the expression of periodontal markers in gingival fibroblasts in vitro.

| Overview of experiments
To investigate the interactions between PDL and gingival cells, firstly PDL and gingival cells were separately labeled with cell tracker fluorescent labels and then co-cultured. PDL and gingival cells were then sorted by flow cytometry and expression of PDL markers determined by qPCR. Further co-culture experiments were then carried out using transwell plates to physically separate PDL and GF cells in the same culture medium. Finally, further experiments were carried out using conditioned medium collected from PDL and GF cells. Preliminary investigation of signaling pathways involved in cell interactions was then carried out by adding a Wnt signaling inhibitor, Dkk1, and a BMP signaling inhibitor, noggin, to GF cultures treated with PDL conditioned medium, and measuring effects on ALP expression.

| Analysis of qRT-PCR data
To calculate relative expression, all samples were normalized housekeeping gene expression (RPL13a) cycle threshold (Ct) value and then normalized to the average of PDL Ct value at baseline day 0 (24 h after seeding the cells). Fold changes of expression of gene of interest were assessed using the comparative 2 −(delta delta Ct) method.
Data are expressed as means ± SD.

| Direct co-cultures
PDL and GF cells were cultured and grown in normal media at 37°C.
After the cells reached confluence, the cells were subcultured with trypsin/EDTA and stained with cell tracker fluorescent markers.
CellTracker TM Green (C7025 Invitrogen) and CellTracker TM Orange (C34551 Invitrogen) were used to label gingival fibroblasts and PDL cells, respectively, in direct co-culture and their single culture controls.
When the cells were approximately 90% confluent, the cells were detached with trypsin after washing with PBS and centrifuged for 5 min at 1100 rpm. Then, the pre-warmed 5 μM CellTracker TM dye working solution was added and incubated for 30 min in CO 2 incubator. The cells were centrifuged and washed by PBS to remove excess dye. After being stained with cell trackers, the cells were divided into two groups, those for FACS sorting and those not for FACS.
PDL cells and GF were directly co-cultured in the same wells of 12-well plates with the ratio of PDL cells to GF of 1:3. As a control group, single cultures of PDL cells and GF were used and seeded at the same amount as the total cell amount of co-culture.
Initial density of PDL cells in co-culture was 5 × 10 3 cells/well, whereas GF was 1. Single cultures with cell tracker staining were used as positive controls, and unstained cells were used to set optimal side and forward scatter voltages on the FACS machine and prevent unwanted cells before cell sorting. Cells were sorted on a 100μm nozzle. All sorted cells were cultured in new 12-well plates as a single culture and after 24 h, the cells were processed for total RNA extraction and real-time PCR. Cells were then cultured until reaching 80% confluence and lysed with TRIzol ® Reagent (Thermo Fisher, Scientific) for processing of total RNA extraction.

| Conditioned medium
PDL cells and GF were grown with normal media in flask T75 with the same initial density of 1 × 10 5 cells at 37°C in a humidified 5% CO 2 , 95% air atmosphere. After the cells reached 90-95% confluence, the normal media were removed and the cells were washed with PBS 3×; then, 5 ml of normal medium without serum was added into the flask. The cells were incubated at 37°C in a humidified 5% CO 2 , 95% air atmosphere for 24 h. Conditioned medium was collected and filtered with 0.22μm strainer and stored frozen at −20°C prior to use in experiments.
PDL and GF cells were seeded at a density 15 000 cells per wells into 12-well plates in triplicates at passage 5. All the cells were grown with normal media (NM), and after reaching 90-95% confluence, medium was removed, the cells were washed with PBS and the collected conditioned medium was supplemented with the addition of 1% serum and then cultured for 3 days before processing to RNA extraction. Additional controls of normal medium supplemented with the addition of 1% serum were also employed.

| Alkaline phosphatase (ALP) assays
Cell culture medium was removed, and cells were washed with 100 μl PBS. After washing, PBS was discarded and 50 μl distilled water was added into the wells. Cell plates were placed in the incubator at 37°C for 15 min and transferred into −80°C for 20 min. This freeze-thaw process was repeated three times.
A serial dilution of p-nitrophenol standard (Sigma-Aldrich) was prepared down to 3.125 μg/ml with the final volume 100 μl.

| Effects of inhibition of Wnt signaling on ALP expression
In preliminary experiments to characterize the nature of the signaling molecules present in conditioned media, we added the Wnt inhibitor Dkk-1 (R&D Systems) to conditioned media at concentrations from 0 to 1000 ng/ml prior to treating GF. The cells were then cultured for 3 days. After 3 days, ALP activity was assessed and results were normalized to results obtained with medium without inhibitor.

| Statistical analysis
All the experiments were carried out in triplicate, and one-way ANOVA with Tukey or Bonferroni post-hoc test was performed for statistical analysis using GraphPad Prism version 5.00 (GraphPad Software, San Diego California). A p-value of <.05 was considered statistically significant.
In On the basis of all these results, GF-3 was selected and used for all subsequent experiments except where indicated.

| Direct co-culture with FACS sorting
Following co-culture for 3 days and subsequent re-sorting into either GF or PDL cells, there was a marked upregulation of all marker genes tested in GF cells when compared to GF monoculture controls ( Figure 2). FACS gating, stained co-cultures, and immunofluorescent detection of asporin, periostin and nestin are shown in Figure S3.
Following co-culture, PDL cells showed down-regulation of asporin.
In addition, it was clear that the process of FACS sorting itself markedly decreased the overall gene expression for all markers tested (eg, asporin expression in GF increased 5.77 fold without sorting compared to 0.73 fold after sorting) ( Figure S4).

| Periodontal marker expression in indirect cocultures and conditioned medium
In order to determine whether interaction between the cell types and ALP expression ( Figure 5D) with GF-3, notable for having the highest percentage of CD105 (Table 2) and both ALP expression and activity levels ( Figure S2). In contrast, GF-4 had shown marked phenotypic differences from the other cell lines that had been ini-

| Alkaline phosphatase activity
ALP protein activity was tested using both co-culture and conditioned medium methods by measuring enzyme activity and histochemical staining.
Histochemical staining was used to test ALP production in direct co-cultures compared to monotype cultures, and the intensity of alkaline phosphatase staining was quantified by plate reader.
Co-cultures treated with osteogenic medium showed a significant increase in alkaline phosphatase activity compared to GF single cultures ( Figure S5). Although enzymatic activity assessment of para-nitrophenol (PNP) release showed that PDL CM consistently elevated ALP activity in GF1-3 cell lines above that of GF CM cultures, this was not statistically different due to the extent of variance between replicates ( Figure 6).  Figure 5D).

| Effect of Dkk-1 on ALP activity in conditioned medium-treated cells
Dkk-1 had no effect on ALP activity in cells grown in normal medium ( Figure 7a). In contrast, addition of Dkk-1 to GF cultures treated with PDL CM resulted in a dose-dependent inhibition of the effect of PDL CM on ALP activity (Figure 7b). In addition, in further experiments with GF cell lines GF-1 and GF-2, PDL CM induced ALP activity, and either addition of 100 ng/ml or 1 µg/ml Dkk-1 significantly decreased the effect of the PDL CM.

| DISCUSS ION
We postulated that periodontal ligament-derived MSCs could in- MSCs have been shown to have a complex secretome which appears to make a major contribution to the properties of MSC such as immunomodulation, homing, cell recruitment, and differentiation. 21,22 There is now considerable interest in the potential therapeutic application of MSC secreted products and the possible exploitation of MSC properties in cell-free applications.

F I G U R E 3
Gene expression of (A) asporin; (B) periostin; (C) nestin; and (D) alkaline phosphatase following indirect co-culture of periodontal cells (PDLSC) and gingival fibroblasts (GFs). PDLSC and GF were grown transwell co-cultures in normal media for 3 days. Single cultures either of PDLSC or GFs acted as control group. Marker expression levels were assessed by qPCR and expression normalized to RPL13a expression. Data represented as mean ± SD. Data were generated from using 3 independent primary PDLSCs lines and 1 independent primary GF line. Three replicates were tested for each cell line. One-way ANOVA with Bonferroni's post-test was conducted for statistical analysis. * p < .05; *** p < .001. Only statistically significant differences indicated . GF were cultured in PDL CM for the test group for 3 days. As a control group, samples were cultured in normal media. All the samples carried out in triplicates and repeated twice. Data shown as mean ± SD. Three replicates were tested for each cell line. One-way ANOVA with Bonferroni's post-test was used for statistical analysis. *** p < .001.
Only statistically significant differences indicated lines we used were isolated in our laboratory by explant culture from the collar of gingival tissue removed with tooth extraction, meaning that the anatomical derivation of these cells is largely from the region of the dentogingival junction.
We have recently shown that there is a clear distinction between periodontal and gingival tissue phenotypes at the dentogingival junction 19 and it is thus possible that our gingival explant cultures represent a variable mixture of gingival and PDL cell phenotypes which would explain the marked heterogeneity between cultures.
These findings further emphasize the general point of the marked heterogeneity of primary gingival fibroblast culture phenotypes.

| CON CLUS ION
Our findings reveal that PDL cells secrete diffusible factors which can induce the expression of PDL markers in gingival fibroblast F I G U R E 7 Effect of Wnt inhibition on alkaline phosphatase (ALP) activity in gingival fibroblasts (GFs) cultured with PDL conditioned medium (PDL CM). GFs were cultured in PDL CM and normal media supplemented with the Wnt inhibitor, Dkk-1. Non-inhibitor samples were used as control. Fold change was calculated relative to non-inhibitor samples in normal media. Data represented as mean ± SD. n = 3 replicates, and the experiment was repeated twice. One-way ANOVA with Bonferroni's post-test was used for statistical analysis. * p < .05; ** p < .01; ***p < .001. Only statistically significant differences indicated